Your browser doesn't support javascript.
loading
Mostrar: 20 | 50 | 100
Resultados 1 - 11 de 11
Filtrar
Mais filtros










Base de dados
Intervalo de ano de publicação
3.
Neuroscience ; 508: 40-51, 2023 01 01.
Artigo em Inglês | MEDLINE | ID: mdl-36464177

RESUMO

Advances in single cell sequencing have enabled the identification of a large number of genes, expressed in many different cell types, and across a variety of model organisms. In particular, the nervous system harbors an immense number of interacting cell types, which are poorly characterized. Future loss- and gain-of-function experiments will be essential in determining how novel genes play critical roles in diverse cellular, as well as evolutionarily adapted, contexts. However, functional analysis across species is often hampered by technical limitations, in non-genetic animal systems. Here, we describe a new single plasmid system, misPiggy. The system is based around the hyperactive piggyBac transposon system, which combines stable genomic integration of transgenes (for long-term expression) with large cargo capacity. Taking full advantage of these characteristics, we engineered novel expression modules into misPiggy that allow for cell-type specific loss- and gain-of-gene function. These modules work widely across species from frog to ferret. As a proof of principle, we present a loss-of-function analysis of the neuronal receptor Deleted in Colorectal Cancer (DCC) in retinal ganglion cells (RGCs) of Xenopus tropicalis tadpoles. Single axon tracings of mosaic knock-out cells reveal a specific cell-intrinsic requirement of DCC, specifically in axonal arborization within the frog tectum, rather than retina-to-brain axon guidance. Furthermore, we report additional technical advances that enable temporal control of knock-down or gain-of-function analysis. We applied this to visualize and manipulate labeled neurons, astrocytes and other glial cells in the central nervous system (CNS) of mouse, rat and ferret. We propose that misPiggy will be a valuable tool for rapid, flexible and cost-effective screening of gene function across a variety of animal models.


Assuntos
Furões , Neuroglia , Animais , Camundongos , Ratos , Axônios/metabolismo , Células Ganglionares da Retina/metabolismo , Sistema Nervoso Central
4.
Sci Adv ; 8(29): eabo0155, 2022 07 22.
Artigo em Inglês | MEDLINE | ID: mdl-35867795

RESUMO

Dynamic contacts are formed between endoplasmic reticulum (ER) and mitochondria that enable the exchange of calcium and phospholipids. Disturbed contacts between ER and mitochondria impair mitochondrial dynamics and are a molecular hallmark of Parkinson's disease, which is also characterized by impaired complex I activity and dopaminergic neuron degeneration. Here, we analyzed the role of cysteine-rich with EGF-like domain (Creld), a poorly characterized risk gene for Parkinson's disease, in the regulation of mitochondrial dynamics and function. We found that loss of Creld leads to mitochondrial hyperfusion and reduced ROS signaling in Drosophila melanogaster, Xenopus tropicalis, and human cells. Creld fly mutants show differences in ER-mitochondria contacts and reduced respiratory complex I activity. The resulting low-hydrogen peroxide levels are linked to disturbed neuronal activity and lead to impaired locomotion, but not neurodegeneration, in Creld mutants. We conclude that Creld regulates ER-mitochondria communication and thereby hydrogen peroxide formation, which is required for normal neuron function.


Assuntos
Drosophila melanogaster , Doença de Parkinson , Animais , Neurônios Dopaminérgicos/metabolismo , Drosophila melanogaster/genética , Drosophila melanogaster/metabolismo , Retículo Endoplasmático/metabolismo , Humanos , Peróxido de Hidrogênio/metabolismo , Mitocôndrias/genética , Mitocôndrias/metabolismo , Doença de Parkinson/genética , Doença de Parkinson/metabolismo
5.
Neuron ; 109(18): 2864-2883.e8, 2021 09 15.
Artigo em Inglês | MEDLINE | ID: mdl-34384519

RESUMO

The molecular and cellular mechanisms underlying complex axon morphogenesis are still poorly understood. We report a novel, evolutionary conserved function for the Drosophila Wnk kinase (dWnk) and its mammalian orthologs, WNK1 and 2, in axon branching. We uncover that dWnk, together with the neuroprotective factor Nmnat, antagonizes the axon-destabilizing factors D-Sarm and Axundead (Axed) during axon branch growth, revealing a developmental function for these proteins. Overexpression of D-Sarm or Axed results in axon branching defects, which can be blocked by overexpression of dWnk or Nmnat. Surprisingly, Wnk kinases are also required for axon maintenance of adult Drosophila and mouse cortical pyramidal neurons. Requirement of Wnk for axon maintenance is independent of its developmental function. Inactivation of dWnk or mouse Wnk1/2 in mature neurons leads to axon degeneration in the adult brain. Therefore, Wnk kinases are novel signaling components that provide a safeguard function in both developing and adult axons.


Assuntos
Proteínas do Domínio Armadillo/biossíntese , Axônios/metabolismo , Proteínas do Citoesqueleto/biossíntese , Proteínas de Drosophila/biossíntese , Evolução Molecular , Morfogênese/fisiologia , Proteínas Serina-Treonina Quinases/biossíntese , Animais , Proteínas do Domínio Armadillo/antagonistas & inibidores , Proteínas do Domínio Armadillo/genética , Linhagem Celular Tumoral , Proteínas do Citoesqueleto/antagonistas & inibidores , Proteínas do Citoesqueleto/genética , Proteínas de Drosophila/antagonistas & inibidores , Proteínas de Drosophila/genética , Drosophila melanogaster , Feminino , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos da Linhagem 129 , Camundongos Endogâmicos C57BL , Gravidez , Proteínas Serina-Treonina Quinases/genética
6.
Neural Dev ; 16(1): 2, 2021 02 02.
Artigo em Inglês | MEDLINE | ID: mdl-33526076

RESUMO

BACKGROUND: The efficient regenerative abilities at larvae stages followed by a non-regenerative response after metamorphosis in froglets makes Xenopus an ideal model organism to understand the cellular responses leading to spinal cord regeneration. METHODS: We compared the cellular response to spinal cord injury between the regenerative and non-regenerative stages of Xenopus laevis. For this analysis, we used electron microscopy, immunofluorescence and histological staining of the extracellular matrix. We generated two transgenic lines: i) the reporter line with the zebrafish GFAP regulatory regions driving the expression of EGFP, and ii) a cell specific inducible ablation line with the same GFAP regulatory regions. In addition, we used FACS to isolate EGFP+ cells for RNAseq analysis. RESULTS: In regenerative stage animals, spinal cord regeneration triggers a rapid sealing of the injured stumps, followed by proliferation of cells lining the central canal, and formation of rosette-like structures in the ablation gap. In addition, the central canal is filled by cells with similar morphology to the cells lining the central canal, neurons, axons, and even synaptic structures. Regeneration is almost completed after 20 days post injury. In non-regenerative stage animals, mostly damaged tissue was observed, without clear closure of the stumps. The ablation gap was filled with fibroblast-like cells, and deposition of extracellular matrix components. No reconstruction of the spinal cord was observed even after 40 days post injury. Cellular markers analysis confirmed these histological differences, a transient increase of vimentin, fibronectin and collagen was detected in regenerative stages, contrary to a sustained accumulation of most of these markers, including chondroitin sulfate proteoglycans in the NR-stage. The zebrafish GFAP transgenic line was validated, and we have demonstrated that is a very reliable and new tool to study the role of neural stem progenitor cells (NSPCs). RNASeq of GFAP::EGFP cells has allowed us to clearly demonstrate that indeed these cells are NSPCs. On the contrary, the GFAP::EGFP transgene is mainly expressed in astrocytes in non-regenerative stages. During regenerative stages, spinal cord injury activates proliferation of NSPCs, and we found that are mainly differentiated into neurons and glial cells. Specific ablation of these cells abolished proper regeneration, confirming that NSPCs cells are necessary for functional regeneration of the spinal cord. CONCLUSIONS: The cellular response to spinal cord injury in regenerative and non-regenerative stages is profoundly different between both stages. A key hallmark of the regenerative response is the activation of NSPCs, which massively proliferate, and are differentiated into neurons to reconstruct the spinal cord. Also very notably, no glial scar formation is observed in regenerative stages, but a transient, glial scar-like structure is formed in non-regenerative stage animals.


Assuntos
Células-Tronco Neurais , Traumatismos da Medula Espinal , Regeneração da Medula Espinal , Animais , Medula Espinal , Xenopus laevis , Peixe-Zebra
7.
Brain Sci ; 10(3)2020 Mar 13.
Artigo em Inglês | MEDLINE | ID: mdl-32183137

RESUMO

Astrocytes are ubiquitous in the central nervous system (CNS). These cells possess thousands of individual processes, which extend out into the neuropil, interacting with neurons, other glia and blood vessels. Paralleling the wide diversity of their interactions, astrocytes have been reported to play key roles in supporting CNS structure, metabolism, blood-brain-barrier formation and control of vascular blood flow, axon guidance, synapse formation and modulation of synaptic transmission. Traditionally, astrocytes have been studied as a homogenous group of cells. However, recent studies have uncovered a surprising degree of heterogeneity in their development and function, in both the healthy and diseased brain. A better understanding of astrocyte heterogeneity is urgently needed to understand normal brain function, as well as the role of astrocytes in response to injury and disease.

8.
J Comp Neurol ; 526(10): 1712-1732, 2018 07 01.
Artigo em Inglês | MEDLINE | ID: mdl-29603210

RESUMO

Studying the cellular composition and morphological changes of cells lining the central canal during Xenopus laevis metamorphosis could contribute to understand postnatal development and spinal cord regeneration. Here we report the analysis of central canal cells at different stages during metamorphosis using immunofluorescence for protein markers expression, transmission and scanning electron microscopy and cell proliferation assays. The central canal was regionalized according to expression of glial markers, ultrastructure, and proliferation in dorsal, lateral, and ventral domains with differences between larvae and froglets. In regenerative larvae, all cell types were uniciliated, have a radial morphology, and elongated nuclei with lax chromatin, resembling radial glial cells. Important differences in cells of nonregenerative froglets were observed, although uniciliated cells were found, the most abundant cells had multicilia and revealed extensive changes in the maturation and differentiation state. The majority of dividing cells in larvae corresponded to uniciliated cells at dorsal and lateral domains in a cervical-lumbar gradient, correlating with undifferentiated features. Neurons contacting the lumen of the central canal were detected in both stages and revealed extensive changes in the maturation and differentiation state. However, in froglets a very low proportion of cells incorporate 5-ethynyl-2'-deoxyuridine (EdU), associated with the differentiated profile and with the increase of multiciliated cells. Our work showed progressive changes in the cell types lining the central canal of Xenopus laevis spinal cord which are correlated with the regenerative capacities.


Assuntos
Metamorfose Biológica , Medula Espinal/citologia , Medula Espinal/fisiologia , Xenopus laevis/anatomia & histologia , Xenopus laevis/fisiologia , Animais , Contagem de Células , Proliferação de Células , Cílios/ultraestrutura , Desoxiuridina/análogos & derivados , Feminino , Larva , Masculino , Regeneração Nervosa , Células-Tronco Neurais , Neuroglia/fisiologia , Neuroglia/ultraestrutura , Medula Espinal/crescimento & desenvolvimento
9.
Nat Protoc ; 12(2): 372-389, 2017 02.
Artigo em Inglês | MEDLINE | ID: mdl-28102835

RESUMO

Here we present a protocol for the husbandry of Xenopus laevis tadpoles and froglets, and procedures to study spinal cord regeneration. This includes methods to induce spinal cord injury (SCI); DNA and morpholino electroporation for genetic studies; in vivo imaging for cell analysis; a swimming test to measure functional recovery; and a convenient model for screening for new compounds that promote neural regeneration. These protocols establish X. laevis as a unique model organism for understanding spinal cord regeneration by comparing regenerative and nonregenerative stages. This protocol can be used to understand the molecular and cellular mechanisms involved in nervous system regeneration, including neural stem and progenitor cell (NSPC) proliferation and neurogenesis, extrinsic and intrinsic mechanisms involved in axon regeneration, glial response and scar formation, and trophic factors. For experienced personnel, husbandry takes 1-2 months; SCI can be achieved in 5-15 min; and swimming recovery takes 20-30 d.


Assuntos
Regeneração da Medula Espinal , Xenopus laevis/fisiologia , Criação de Animais Domésticos , Animais , Feminino , Masculino , Células-Tronco Neurais/citologia
10.
Dev Biol ; 408(2): 229-43, 2015 Dec 15.
Artigo em Inglês | MEDLINE | ID: mdl-25797152

RESUMO

Spinal cord regeneration is very inefficient in humans, causing paraplegia and quadriplegia. Studying model organisms that can regenerate the spinal cord in response to injury could be useful for understanding the cellular and molecular mechanisms that explain why this process fails in humans. Here, we use Xenopus laevis as a model organism to study spinal cord repair. Histological and functional analyses showed that larvae at pre-metamorphic stages restore anatomical continuity of the spinal cord and recover swimming after complete spinal cord transection. These regenerative capabilities decrease with onset of metamorphosis. The ability to study regenerative and non-regenerative stages in Xenopus laevis makes it a unique model system to study regeneration. We studied the response of Sox2(/)3 expressing cells to spinal cord injury and their function in the regenerative process. We found that cells expressing Sox2 and/or Sox3 are present in the ventricular zone of regenerative animals and decrease in non-regenerative froglets. Bromodeoxyuridine (BrdU) experiments and in vivo time-lapse imaging studies using green fluorescent protein (GFP) expression driven by the Sox3 promoter showed a rapid, transient and massive proliferation of Sox2(/)3(+) cells in response to injury in the regenerative stages. The in vivo imaging also demonstrated that Sox2(/)3(+) neural progenitor cells generate neurons in response to injury. In contrast, these cells showed a delayed and very limited response in non-regenerative froglets. Sox2 knockdown and overexpression of a dominant negative form of Sox2 disrupts locomotor and anatomical-histological recovery. We also found that neurogenesis markers increase in response to injury in regenerative but not in non-regenerative animals. We conclude that Sox2 is necessary for spinal cord regeneration and suggest a model whereby spinal cord injury activates proliferation of Sox2/3 expressing cells and their differentiation into neurons, a mechanism that is lost in non-regenerative froglets.


Assuntos
Fatores de Transcrição SOXB1/fisiologia , Regeneração da Medula Espinal/fisiologia , Proteínas de Xenopus/fisiologia , Xenopus laevis/crescimento & desenvolvimento , Xenopus laevis/fisiologia , Animais , Animais Geneticamente Modificados , Proliferação de Células , Regulação da Expressão Gênica no Desenvolvimento , Técnicas de Silenciamento de Genes , Humanos , Larva/crescimento & desenvolvimento , Larva/fisiologia , Metamorfose Biológica , Modelos Animais , Modelos Neurológicos , Neurogênese , Fatores de Transcrição SOXB1/antagonistas & inibidores , Fatores de Transcrição SOXB1/genética , Traumatismos da Medula Espinal/genética , Traumatismos da Medula Espinal/patologia , Traumatismos da Medula Espinal/fisiopatologia , Regeneração da Medula Espinal/genética , Proteínas de Xenopus/antagonistas & inibidores , Proteínas de Xenopus/genética , Xenopus laevis/genética
11.
Genesis ; 51(8): 529-44, 2013 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-23760835

RESUMO

Unlike mammals, regenerative model organisms such as amphibians and fish are capable of spinal cord regeneration after injury. Certain key differences between regenerative and nonregenerative organisms have been suggested as involved in promoting this process, such as the capacity for neurogenesis and axonal regeneration, which appear to be facilitated by favorable astroglial, inflammatory and immune responses. These traits provide a regenerative-permissive environment that the mammalian spinal cord appears to be lacking. Evidence for the regenerative nonpermissive environment in mammals is given by the fact that they possess neural stem/progenitor cells, which transplanted into permissive environments are able to give rise to new neurons, whereas in the nonpermissive spinal cord they are unable to do so. We discuss the traits that are favorable for regeneration, comparing what happens in mammals with each regenerative organism, aiming to describe and identify the key differences that allow regeneration. This comparison should lead us toward finding how to promote regeneration in organisms that are unable to do so.


Assuntos
Regeneração Nervosa , Medula Espinal/fisiologia , Animais , Axônios/fisiologia , Humanos , Mamíferos , Neurogênese , Traumatismos da Medula Espinal/imunologia , Traumatismos da Medula Espinal/patologia , Traumatismos da Medula Espinal/fisiopatologia
SELEÇÃO DE REFERÊNCIAS
DETALHE DA PESQUISA
...